Recombinant forms of glargine have been produced in various microbial expression systems, wherein organisms such as E. coli, Saccharomyces cerevisiae have been employed for the commercial production of recombinant human insulin and derivatives thereof. Owing to certain disadvantages of these systems such as low expression levels, difficulties in downstream purification etc. the use of methylotrophic yeast Pichia pastoris (P. pastoris) has been favored as a protein expression system. The expression system offers several advantages such as high expression, simple processing, low production cost, high density culture (U.S. Pat. No. 6,800,606).
Insulin Glargine is a slow acting insulin analogue. Use of E. coli as an expression system for the expression is already there in the prior art. As E. coli does not have the cellular machinery for folding the expressed polypeptide and establish the disulphide bridges correctly, so there is a need in the art to overcome such folding problem.
The glargine downstream process involves the clipping of the precursor using trypsin. Trypsin has the specificity of clipping at the carboxyl terminal of both ‘K” and ‘R’ (as shown in fig: 1). This results in the generation of more product related impurities (as shown in fig: 1)
1) FVNQHLCGSHLVEALYLVCGER, 2) FVNQHLCGSHLVEALYLVCGERGFFYTPK, 3) FVNQHLCGSHLVEALYLVCGERGFFYTPKTR, 4) GFFYTPKTR 5) TRR
The disadvantages associated with the known downstream processes of the prior art have been remedied in the instant disclosure.
U.S. Pat. No. 4,929,553 and its related applications are concerned with the specific processing of secreted proteins in genetically modified yeast cells. Specifically, this disclosure is concerned with the use of recombinant DNA to produce Kex2 in greater quantities. The expression of proteins and use of Kex2p to processes after the cleave after dibasic amino acid is known in the prior art.
WO2008037735 and its related applications disclosed a method for making mature human insulin or an analogue wherein C-terminal amino acid residue in the B-chain cleaved off by means of a carboxypeptidase activity either within the fungi cell or subsequently in the culture medium.
Hence, there exists a need in the art to produce a process of producing a functional two chain glargine into the medium to enable processing of the insulin glargine into active two chain fully folded form invivo.